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Specific nest type PCR detection reagent kit for clinical pentatrichomonas hominis infection

A detection kit and technology for Trichomonas, applied in the field of molecular biology detection, can solve the problems of complex components, non-specific bands, false positive results, etc., and achieve high sensitivity, good specificity, and reduce false positives.

Inactive Publication Date: 2020-06-02
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complex composition of stool samples, which contain a large number of bacteria, non-specific bands or false positive results often appear when using ordinary PCR methods for detection

Method used

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  • Specific nest type PCR detection reagent kit for clinical pentatrichomonas hominis infection
  • Specific nest type PCR detection reagent kit for clinical pentatrichomonas hominis infection
  • Specific nest type PCR detection reagent kit for clinical pentatrichomonas hominis infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] A specific nested PCR detection kit for clinically using Trichomonas human infection, including two pairs of nested PCR primers, dNTP, PCR buffer, rTaq enzyme, Mg 2+ solution and dd H 2 O; where,

[0041] 1) PCR reaction solution:

[0042] dNTP Mixture (2.5mM / each) 4μL;

[0043] 10×PCR Buffer (Mg 2+ free) 5 μL;

[0044] MgCl 2 (25mM) 8μL;

[0045] DNA template 1 μL;

[0046] r Taq Polymerase (5U / μL) 0.25μL;

[0047] Add ddH 2 O 50 μL;

[0048] 2) 1.0 μL each of two pairs of nested PCR primers is:

[0049] F1: 5'- ATG GCG AGT GGT GGA ATA -3';

[0050] R1: 5'-CCC AAC TAC GCT AAG GAT T-3';

[0051] F2: 5'-TGT AAA CGA TGC CGA CAG AG-3';

[0052] R2: 5'-CAA CAC TGA AGC CAA TGC GAG C-3'.

[0053] Upstream primer (15pM) 1.0μL;

[0054] Downstream primer (15pM) 1.0μL;

[0055] 3) Control: the DNA template of the positive control is the genomic DNA of Pentatrimonas human;

[0056] Negative control is ddH 2 O, for comparing PCR products and monitoring for conta...

Embodiment 2

[0057] Example 2 Nested PCR Primer Design

[0058] Species-specific diagnostic sequence of P. hominis: select a highly conserved 18S rRNA gene partial sequence, and compare it in NCBI to be specific to P. hominis, meeting the criteria for species-specific detection and research. Design specific diagnostic primers according to the specific sequence of human Pentatrichomonas as follows:

[0059] Primers for the first round of PCR: F1: 5'- ATG GCG AGT GGT GGA ATA -3';

[0060] R1: 5'-CCC AAC TAC GCT AAG GAT T-3';

[0061] The second round of PCR primers: F2: 5'- TGT AAA CGA TGC CGA CAG AG-3';

[0062] R2: 5'- CAA CAC TGA AGC CAA TGC GAG C-3';

[0063] Among the above two pairs of primers, F1-R1 is the first-round PCR primer, and F2-R2 is the second-round PCR primer, which can amplify a 339bp diagnostic gene fragment with bright target bands and no non-specific bands. The purpose of sensitively and specifically diagnosing pentatrimonas can be realized.

Embodiment 3

[0064] Example 3 Optimization of PCR reaction conditions

[0065] The optimization process of nested PCR reaction conditions of the present invention is:

[0066] Optimization of the first round of PCR annealing temperature: perform temperature gradient PCR amplification, set the annealing temperature to 52.0°C, 52.6°C, 54.3°C, 55.0°C, 56.5°C, 57.8°C, 58.4°C, 59.5°C, 60.3°C, 62.0°C , the results showed that the optimum annealing temperature was 55.0°C (see attached figure 1 a).

[0067] The second round of PCR annealing temperature optimization: Carry out temperature gradient PCR amplification, set the annealing temperature to 55.0°C, 56.3°C, 57.5°C, 58.2°C, 59.6°C, 60.5°C, 61.4°C, 63.0°C, the results show the best annealing temperature is 55.0°C (see attached figure 1 b).

[0068] Mg 2+ Optimization of Concentration: Setting Mg 2+ The concentration gradients were 0.125 mmol / L, 0.25 mmol / L, 0.375 mmol / L, 0.5 mmol / L, 1 mmol / L, 1.5 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5...

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Abstract

The invention provides a specific nest type PCR detection reagent kit for clinical pentatrichomonas hominis infection. Through two times of PCR amplification, the reagent kit enables a detection method to have higher sensitivity, and is used for specific sensitive fast detection of pentatrichomonas hominis infection in dung (or polluted water samples) of people or animals clinically; the reagent kit has favorable specificity, and misjudgment of detection samples is reduced; and the reagent kit has extremely high application value in the respect of prevention, control and early warning of dungpollution.

Description

technical field [0001] The present invention provides a specific nested PCR detection kit for clinically using Pentatrichomonas human infection, which is used for the detection of Pentatrichomonas human infection in other populations (or animal feces or polluted water samples) such as tumor patients. A preparation method for the above-mentioned PCR detection is disclosed, which belongs to the technical field of molecular biology detection. Background technique [0002] Trichomonas hominis ( Pentatrichomonas hominis ), is an important zoonotic parasite, which can reproduce in the large intestine of animals such as humans, dogs, pigs, cats, non-human primates and rodents, and is excreted through feces. Direct infection, clinical tumor patients have a higher infection rate. The detection methods of P. hominis that have been reported at home and abroad include smear microscope inspection, culture method, common PCR detection method, in situ hybridization detection method and s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6848
CPCC12Q1/6848C12Q2549/119
Inventor 张西臣张洪波张楠宫鹏涛李建华杨举李赫李新
Owner JILIN UNIV
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